The activation of the complement system can be achieved by three different pathways, the classical antibody-dependent activation pathway, the alternative activation pathway and the lectin-activation pathway. The alternative activation pathway as well as the lectin-pathway were shown not to be dependent on antibodies. All pathways share a similar cascade-like organization, wherein a protease acts on zymogenes of a subsequent protease. This cascade results in an amplification of the initiation signals. The central step of the complement cascade resides in the formation of a C3-convertase, which cleaves C3 to C3b and C3a (FIG. 1). Subsequently, the resulting C3b can act as a part of a C5-convertase, which cleaves C5 in C5b and C5a. In the terminal pathway, the gradual accumulation of C6, C7, C8 and several molecules C9 results in the formation of the membrane attack complex which is capable of forming a pore in the membrane of the target cells, thereby effecting lysis of the cells.
The complement protein C3 is the central component of all activation pathways. It is predominantly expressed in the liver as a 1663 amino acid precursor protein (Alper et al., 1969). After the 22 amino acid signal sequence has been cleaved off, the precursor protein is proteolytically cleaved into two chains by removal of four arginine residues. The resulting α-chain has a molecular weight of 115 kDa and the β-chain has a molecular weight of 73 kDa (DeBruijn and Fey, 1985). The chains are linked by a disulfide bridge and by non-covalent interactions (Dolmer and Sottrup-Jensen, 1993; Janatova, 1986). Furthermore, the resulting 188 kDa protein carries a carbohydrate chain on each chain, which consists of 5 to 9 mannose residues and two N-acetylglucosamine residues (Hirani et al., 1986).
C3 is cleaved between the amino acids Arg726 and Ser727 by the C3-convertases. The 9 kDa C3a, which results from the cleavage, is an anaphylatoxin and causes an increase in chemotaxis as well as an increase in the permeability of the blood capillaries. By cleavage of the 179 kDa-C3b between the amino acid Cys988 and Glu991 a highly reactive thioester is released, by the use of which C3b binds on the cell surfaces via transacetylation (Tack et al., 1980). Furthermore, several binding sites for different complement proteins are exposed by the cleavage, which explains the various interactions of the C3,-b molecule. Several regulatory complement proteins interact with C3b, which comprises binding sites for CR1 or Factor H, which act as co-factors for the cleavage by Factor I. Factor I cleaves C3b between Arg1281 and Ser1282, and Arg1298 and Ser1299, whereby the fragments C3f and C3bi emerge, the latter of which is inactive and unable to bind Factor B and C5 (Lachmann et al., 1982; Davis et al., 1982). C3bi, however, is capable to remain on the surface of pathogens, where it is recognized by CR3, which occurs on macrophages and killer cells. Subsequently, CR3 mediates the destruction of pathogens (Newman et al., 1984). In case CR1 acts as a co-factor for the protease, Factor I can additionally cleave between amino acids Arg932 and Glu933, thereby forming C3dg and C3c (Ross et al., 1982). C3dg is also capable to remain on the surface and is recognized by CR2 (CD21), which is expressed on B-lymphocytes and dendritic cells (Law and Dodds, 1997). The binding of C3dg to the complement receptor CR2 leads to the activation of B cells (Bohnsack and Cooper, 1988).
After the degradation of C3 by a protease from the venom of the cobra Naja siamensis the fragment C3o is formed, which no longer contains the amino acids 730-739. However, C3o is capable of binding Factor B (O'Keefe et al., 1988). In contrast, the cleavage product of the Factor I proteolysis C3c cannot form a convertase. Based on the comparison of C3c and C3o, one region in C3o of the amino acid sequence 933EGVQKEDIPP appeared to be responsible for binding to Factor B. In further studies, the amino acids 937KED were mutated to alanine. However, no changes in the binding characteristics of Factor B to C3b could be shown (Taniguchi-Sidle und Isenman, 1994).
Activated complement proteins cannot distinguish between external substances and substances which occur naturally in the body. Thereby, it is ensured that for example self-reactive B-cells can be eliminated. Thus, a plurality of regulatory mechanisms is necessary for protecting healthy cells which occur naturally in the body.
The regulation is effected by short half life of the activated complement proteins on the one hand and by plasma proteins such as the C1-Inhibitor (C1-Inh), Factor H and Factor I, as well as membrane-bound proteins such as the Decay-Accelerating-Factor (DAF, CD55), the Membrane-Cofactor-Protein (MCP, CD46) and the Complement Receptor 1 (CR1, CD35) on the other hand, which regulate the complement cascade on specific levels.
C1-Inh controls the activation of C1 by binding to activated C1r and C1s which results in the dissociation of C1q. The time period for the cleavage of C2 and C4 by activated C1 is restricted to a few minutes by C1-Inh (Mollnes und Lachmann, 1988). The C4-binding protein (C4bp) binds to C4b and separates it from C2b. Additionally, it acts as a co-factor for the cleavage of C4b and C3b by Factor I (Scharfstein et al., 1978). The C3-convertase of the classical pathway is inactivated in the same manner by DAF, which exists on all peripheral cells of the blood, epithet and endothel (Lublin and Atkinson, 1989, Lublin and Atkinson, 1990).
C3b represents the central component of all three activation pathways. C3b is regulated by Factor H, CR1, DAF as well as by MCP. Here, Bb is competitively displaced by CR1, Factor H and DAF from the complex of the C3-convertase C3bBb (Makrides et al., 1992). Subsequently, C3b is cleaved by Factor I and inactivated (Pangburn and Müller-Eberhard, 1984). MCP directly attacks C3b and is also a co-factor for the cleavage by Factor I. Protectin (CD95) is a further membrane-bound regulatory protein. It inhibits the polymerization of C9 by binding to C8 and C9 (Mollnes and Lachmann, 1988).
Besides the regulation for the activation, an additional transcriptional control of the complement genes exists. For example, several genes of the complement proteins are upregulated by cytokine and IFNγ-activated transcription factors after damaging a tissue (Volanakis, 1995).
The strict regulatory mechanisms prevent an attack of the complement system on cells which occur naturally in the body. However, body tissue can be damaged by unregulated activation triggered by diverse diseases. In this situation, the activation of the complement is not the primary reason for disease. However, the resulting damaging of the tissue is mediated by the complement. Diseases which are connected with the activation of the complement can be divided into three groups: Chronical diseases, acute diseases and incompatibility towards biomaterials. The group of acute diseases comprise for example asthma (Regal et al., 1993; Regal and Fraser, 1996), sepsis (Hack et al., 1989; Hack et al., 1992), hyperacute rejection in connection with transplantations or xenotransplantations (Bach et al., 1995; Baldwin et al., 1995), pneumonia (Eppinger et al., 1997) and cardiac infarction (Kilgore et al., 1997), as well as a massive C3a-accumulation, which occurs in connection with the cardiopulmonale bypass-operation (Kirklin et al., 1983; Homeister et al., 1992). The chronical diseases comprise, for example, systemic lupus erythematodes (SLE) (Belmont et al., 1986; Buyon et al., 1992), glomerulonephritis (Couser et al., 1985; Couser et al., 1995), rheumatoide arthritis (Kemp et al., 1992; Wang et al., 1995), Alzheimer's disease (Rogers et al., 1992; Morgan et al., 1997), myastenia gravis (Lennon et al., 1978; Piddlesden et al., 1996) and multiple sclerosis (Piddlesden et al., 1994; Williams et al., 1994) as well as organ rejection after transplantations or xenotransplantations (Baldwin et al., 1995; Dalmasso, 1997). The group of incompatibilities towards biomaterials was described in connection with operation material at a cardiopulmonal bypass (Craddock et al., 1977; Mollnes, 1997), with depositions of blood platelets (Gyongyossy-Issa et al., 1994) and with conducting hemodialysis (Cheung et al., 1994; Mollnes, 1997).
A reduced protein concentration of a complement protein or mutations which lead to a total loss of the protein are the reason for many complement-associated diseases. Factor I-deficiency results in a very small content of C3 and other complement proteins of the cascade in the blood. This leads to diverse diseases, such as a monthly occuring meningitis which is associated with menstruation (Gonzales-Rubio et al., 2001). Factor H-deficiency by gene mutation is associated with the hemolytic-uremic syndrom (Zipfel et al., 2001). An unrestricted activity in the classical activation by depletion of C1, C2 or C4 leads for example to a higher disposition towards systemic lupus erythematodes (Morgan and Walport, 1991). A depletion of a component from the alternative activation such as Factor B or Factor D leads to a higher susceptibility towards infections (Morgan and Walport, 1991).
Complement-associated diseases occur both with an increased and decreased complement activation. In case the regulation is disturbed or the activation is prevented, effetive complement modulators are needed.
The group of complement inhibitors for therapeutic use comprises proteins such as the C1-Inhibitor and the soluble complement regulators sCR1 (soluble CR1), sMCP or sDAF, antibodies against C5 or C3 and smaller molecules such as the peptide Compstatin or RNA-aptamers. Several complement inhibitors are tested in clinical phases I, II or III, such as the C5-Inhibitor Pexelizumab, a monoclonal antibody for use at cardiopulmonal bypass (Whiss, 2002) or the soluble complement receptor sCR1 (Zimmerman et al., 2000).
The C1-Inhibitor is the only plasma protein which has been tested in in vivo-studies (Struber et al., 1999; Horstick, 2002). The serine protease is a suicide inhibitor of the serpine family which inhibits activated C1s and C1q by binding to the active site (Sim et al., 1979). The disadvantages of these molecules relate to the sole inhibition of the classical activation pathways as well as in the susceptibility of the protein towards the inactivation by elastase. For this reason, elastase-resistent C1-Inhibitor mutants were generated (Eldering et al., 1993).
The recombinant complement inhibitors embrace soluble regulators such as sCR1, sMCP and sDAF (Christiansen et al., 1996). The soluble complement receptor sCR1 acts as C3- and C5-convertase-inhibitor and has been tested successfully in diverse animal models such as for myasthenia gravis (Piddlesden et al., 1996), multiple sclerosis (Piddlesden et al., 1994) or asthma (Regal et al., 1993). By altering the conditions of expression, it was possible to increase the short half-life of approx. 8 h in vivo up to 70 h. It is proposed that a different glycosylation pattern is responsible for the increased half-life (Weismann et al., 1990; Zimmerman et al., 2000).
The complement receptors MCP and DAF act as complement inhibitors both in vitro and in vivo, for example in the model of reverse passive Arthus-reaction (Moran et al., 1992; Christiansen et al., 1996). sDAF accellerates the decomposition of both the classical and the alternative C3- and C5-convertases. However, sDAF does not act as a co-factor for the cleavage of Factor I (Kinoshita et al., 1985). In contrast, sMCP acts as co-factor for the cleavage of C3b and C4b by Factor I. However, it does not act on the convertases (Liszewski and Atkinson, 1992).
Protectin (CD59) is a further membrane protein which protects naturally occuring cells of the body from MAC-mediated damage. It binds to C5b-8 and prevents the formation of a pore in the membrane by binding of C9 (Davies, 1996). Its soluble counter-part, sCD59, showed inhibition in vitro (Sugita et al., 1994).
A further group of complement inhibitors consists of antibodies, wherein C5 in particular represents an attractive target protein, since its concentration in the serum is clearly lower than the one of C3. Monoclonal antibodies combine the advantage of specifity and high affinity with a relatively long half-life and the ease of production in large amounts. One prerequisite for the therapeutic application is the human origin of the antibodies which prevents an immune response, for example the human anti-mouse-antibody-response. Several antibodies against C3 (Kemp et al., 1994), C3a (Burger et al., 1988; Elsner et al., 1994) or against C5a (Ames et al., 1994; Park et al., 1999) have been developed. Some have been tested in different animal models, for example for nephritis (Wang et al., 1996), collagen-induced arthritis (Wang et al., 1995), myocardial ischemia und reperfusion (Vakeva et al., 1998).
Anaphylatoxin-receptor-antagonists (Konteatis et al., 1994; Pellas et al., 1998; Heller et al., 1999) and RNA-aptamers, which inhibit the C5-cleavage (Biesecker et al., 1999) belong to the group of complement inhibitors with low molecular weight. Compstatin, a C3-Inhibitor, binds to native C3 and prevents its cleavage in C3b. By application of Compstatin, the hyperacute rejection of transplants in an ex-vivo pig-to-human-liver-transplantation was prevented (Fiane et al., 1999a; Fiane et al., 1999b).
Cobra Venom Factor (CVF) is a potent complement modulator of natural origin. CVF is a 149 kDa-glycoprotein from the venom of the cobra species Naja, Ophiophagus and Hemachatus (Müller-Eberhard and Fjellström, 1971). The non-toxic protein consists of three chains, the 68 kDa α-chain, the 48 kDa β-chain and the 32 kDa γ-chain which are linked by disulfide bridges. Additional intramolecular disulfide bridges exist both in the α- and β-chain (one in the α-chain and six in the β-chain; Vogel et al., 1996). The γ-chain can exhibit different sizes due to different processing on the C-terminus (Vogel and Müller-Eberhard, 1984). Two carbohydrate residues are attached to the α-chain and one to the β-chain in form of complex, N-bound oligosaccharide chains (Vogel and Müller-Eberhard, 1984; Grier et al., 1987).
The percentual composition of the secondary structure of CVF was determined by circular dichroism. The composition shows a high analogy to the composition of the secondary structures of the human three-chain C3-derivate C3c. For CVF 11% helices, 47% β-sheets and 18% β-loops were determined. The C3c-molecule also has 11% helices and 47% β-sheets. In contrast, human C3 consists of 24% helices and 32% β-sheets (Vogel et al., 1984). In the primary structure of the pre-pro-CVF the α-chain is encoded first, followed by the γ-chain and subsequently by the β-chain. On the C-terminus of the α-chain 4 arginine residues are located, followed by aC3a-homologous region. Subsequent to the γ-chain, a C3d-homologous region is located. Both the signal peptide and the arginine residues and the C3a- and C3d-homologous regions are removed post-translationally, thereby generating the three-chain structure. The venom protease, which is thought to be responsible for the modification also cleaves C3 in a CVF-similar structure (O'Keefe et al., 1988).
CVF shares an identity of 85% and a similarity of 92% on the protein level with cobra C3 (coC3). With human C3 the identity amounts to 51% and the similarity to 70% (Fritzinger et al., 1992; Fritzinger et al., 1994; Vogel et al., 1996). Moreover, both proteins have a chain structure of the same kind.
This high similarity is also reflected by the fact that CVF—as C3b— can bind to Factor B and forms a convertase by the Factor D-initiated cleavage of B in Bb and Ba. In contrast to C3Bb, the CVF-dependent convertase CVFBb is a C3- and C5-convertase. By the resistence of CVFBb towards Factor H and Factor I, a convertase is formed with a much higher half-life of 7 h (Vogel and Müller-Eberhard, 1982) under physiological conditions. In comparison, C3bBb has a half-life of 1.5 min (Medicus et al., 1976).
In addition to the increased stability, the CVF-dependent convertase CVFBb cleaves C3 and C5 also in fluid phase, whereas the dependent convertase C3bBb is only active when bound to the cell surface (Vogel et al., 1996). CVF unifies all the above characteristics and leads to a permanent activation of the complement system and to decomplementation resulting thereof.
The decomplementing characteristic of CVF offers a variety of applications. After decomplementation the synthesis of complement protein takes approx. 7 days; during this time e.g. the function of the complement system in the immune response in vivo as well as in the pathogenesis of diseases can be studied (Cochrane et al., 1970; Ryan et al., 1986).
In various xenotransplantation models, such as liver transplantation from guinea pigs to rats, heart transplantations from hamsters to mice as well as islet cell transplantations from rats to mice, CVF was successfully employed (Chrupcala et al., 1994; Chrupcala et al., 1996; Lin et al., 2000; Oberholzer et al., 1999) In all these cases, hyperacute rejection of transplant could be prevented by CVF. Different studies demonstrate that also for diseases like arthritis (Lens et al., 1984), arteriosclerosis (Pang and Minta, 1980) and encephalomyelitis (Morariu and Dalmasso, 1978) CVF can be therapeutically employed.
The problem with therapeutic applications of CVF, however, predominantly resides in the strong immunogenic character of CVF. CVF contains a foreign peptide structure and complex, N-bound oligosaccharide chains with terminal galactosyl residues, which have a significant immunogenic potential (Taniguchiet al., 1996). Consequently, CVF is not suitable for repetitive application. With a relative high portion of carbohydrate structures (7.4%, Vogel and Müller-Eberhardt, 1984) CVF differs clearly from human C3 which only has 1.7% (Hirani et al., 1986). Activity analyses in complement consumption-assays and bystander lysis-assays of CVF deglycosylated by n-glycanase showed that the oligosaccharide chains of CVF are not necessary for both C3-convertase and C5-convertase activity. A reduction of the immunogenicity, however, cannot be achieved by deglycosylation since deglycosylated CVF is still strongly immunogenic due to its foreign amino acid composition.
In an attempt to reduce the immunogenicity of CVF, the CVF α-chain was replaced by the corresponding human C3-β-chain (Kölln et al., 2001). The resulting hybrid protein, however, is still strongly immunogenic and thus inappropriate for therapeutic uses.
From these published results, no information is available which could aid in designing a human C3-derivative i) capable of forming a stable C3-convertase comparable to CVFBb, ii) and suitable for therapeutic applications. Furthermore, published CVF/cobra C3 hybrids (Wehrhahn, 2000) are not suitable to provide any valuable data with regard to the tertiary structure of a C3-derivative required for effective binding of Factor B and for increasing the half life of the resulting C3-convertase.
The identity of cobra C3 with human C3 is too low to allow any specific structural conclusions.
Accordingly, there exists a need to identify polypeptides that exhibit complement-depleting activity and to develop methods of preparing these compounds recombinantly as therapeutics. There also exists a need to identify polypeptides having reduced or eliminated immunogenicity, which polypeptides can be used therapeutically for treating complement-associated disorders and disorders affected by complement activation, respectively.
The present invention satisfies this need, and provides related advantages as well.